Abstract of administrative supplement The original objective was to use a novel plant biotechnology, ?target-directed evolution?, to produce novel inhibitors of the human dopamine transporter (DAT) as leads for medications for alcohol use disorder. This approach evolves plant biosynthesis toward a specific target by a combination of mutagenesis and selection. Lobelia cardinalis was used as the plant species because it contains lobinaline, a previously uninvestigated inhibitor of the DAT that also has partial agonist activity at nicotinic receptors [Brown et al 2015]. These proteins are molecular targets in alcohol and nicotine use disorders, and lobinaline inhibits alcohol consumption in rodents with no evidence of abuse potential. Lobinaline would be an excellent lead compound for pharmacotherapy of alcohol and nicotine use disorders except that it is a highly complex, insoluble alkaloid with no chemical synthesis. However, target-directed evolution of L. cardinalis mutants should generate lobinaline derivatives with greater potency and/or solubility, as well as other metabolites with activity on the DAT. This was shown to be true using a combination of GC/MS and pharmacological analyses [Brown et al 2016]. However, to definitively identify the active metabolites in mutant plant cell cultures the applicants had to develop novel analytical methodology. This has identified at least 15 novel metabolites that are markedly overproduced in those mutants that contain increased inhibitory activity on the DAT. Two of these are already known to be active, either against the DAT or against MPP+ neurotoxicity, and four metabolites are derivatives of lobinaline. These lobinaline metabolites are N-oxides, and derivatives with new double bonds in the ring structure. The lobinaline N-oxides have been tested in vitro and in vivo with exciting results. Their pharmacology in vitro differs only modestly from lobinaline, but they have an important ?druggable? advantage in increased aqueous solubility. However, when tested in vivo by microperfusion in rat brain the lobinaline N-oxides have clear initial inhibitory effects on rate of dopamine clearance (as does lobinaline), but this is rapidly converted to an increase in dopamine clearance capacity. This ?partial antagonist? effect of these metabolites on the DAT is unlike lobinaline, or indeed any other known DAT inhibitor. It is theoretically of considerable value in drug dependence, but this has not been tested in intact animals (and this was not included in the original application). One specific aim in this supplementary proposal is to test the lobinaline N-oxides in animal models of alcohol and nicotine use disorder, and to test the 9 other novel metabolites for activity in vitro. In addition, the mutant transgenic cultures that contain novel active metabolites are a major resource (estimated cost to date is $10K/clone) and for sufficient metabolites to be obtained for testing they must continue to be maintained and sub-cultured. If this supplemental application is successful the applicants intend to use the data for another NIH application to continue development of these novel plant metabolites as treatments for alcohol use disorder.